COMPARISON OF GRADIENT SLOPE, COLUMN LENGTH, AND …We would like to thank Mike Lavallee and Bob...
1
TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS ©2006 Waters Corporation OVERVIEW Purpose: ♦ Directly compare performance of 75 μm i.d. nanocolumns packed with 1.7 μm versus 3 μm particles for peptide digest separations, investigating effects of column length and gradient length (slope) on peak capacity Methods: ♦ 10,000 psi nanoACQUITY UPLC™ system coupled to a quadrupole time-of-flight (Q-Tof) instrument Results: ♦ 1.7 μm particle columns yield higher overall peak capacity gains with column and gradient length vs. 3 μm particle columns ♦ Shorter (ex. 15 cm) 1.7 μm columns are capable of yielding higher efficiencies vs. longer (ex. 50 cm) 3 μm columns ♦ There is a tradeoff between increased peak capacity and slight reduction in MS signal intensity with longer gradient times COMPARISON OF GRADIENT SLOPE, COLUMN LENGTH, AND PARTICLE SIZES FOR ANALYSIS OF PEPTIDES ON A 10,000 PSI NANOLC/MS/MS PLATFORM Jeffrey W. Finch , Hongji Liu, Keith Fadgen, Geoff Gerhardt, James P. Murphy, and John C. Gebler Life Sciences R&D, Waters Corporation, Milford, MA, USA Figure 1. Bridged-ethyl hybrid (BEH) 1.7 μm particle chemistry References 1. Gilar, M.; Daly, A.E.; Kele, M.; Neue, U.D.; Gebler, J.C. J. Chromatogr A 2004, 1061, 183-192. 2. Shen Y.; Zhang, R.; Moore, R.J.; Kim, J.; Metz, T.O.; Hixson, K.K.; Zhao, R.; Livesay, E.A.; Udeseth, H.A.; Smith, R.D. Anal. Chem. 2005, 77, 3090-3100 3.Tolley, L.; Jorgenson, J.W.; and Moseley, M.A. Anal. Chem. 2001, 73, 2985-2991. 4.Finch J.W.; Stapels, M.D.; Fadgen, K.; Liu, H.; Gerhardt, G.; Murphy, J.P.; Ciavarini, S.; Benevides, C.C.; Gebler, J.C. Proceedings of the 53rd ASMS Conference on Mass Spectrometry, San Antonio, TX, June 2005. RESULTS CONCLUSIONS ♦ The nanoACQUITY UPLC system is capable of generating accurate, reliable gradients at pressures approaching 10,000 psi, with good retention time reproducibility over extended periods of run time ♦ Using a 1.7 μm BEH vs. 3 μm particle for a 15 cm column with a 144 min gradient length results in a 59% increase in peak capacity ♦ For short gradient lengths (24 and 48 min), increasing column length does not significantly increase peak capacity for either particle ♦ The 15 cm, 1.7 μm BEH column yields greater peak capacity measured at the various gradient lengths than the significantly longer 50 cm column packed with 3 μm particles ♦ The 30 cm, 1.7 μm BEH column exhibits the greatest overall increase in peak capacity with gradient length ♦ There is a trade-off between increased separation efficiency and slight reduction in MS signal height with longer gradient lengths ♦ Combining the 1.7 μm BEH particles with longer column lengths and longer gradient lengths can provide greater information for nanoLC/MS/MS analysis of complex proteomic samples INTRODUCTION The ability to increase nanoLC separation efficiency is highly desired for obtaining as much MS/MS information as possible where amounts of complex proteomics samples are often limited (1). Methods for improving separation efficiency for typical reversed-phase nanoLC separations include using smaller particles, longer columns, and longer (more shallow) gradient times (2,3). The use of smaller particles (< 2 μm) requires a nanoLC system capable of operating at higher backpressure for extended periods of time (4). However, there have been few fundamental studies comparing effects of particle size, column length, and gradient length (time) on peak capacity, for nanocolumns where a) columns are prepared under very tightly-controlled conditions (same column assembly protocol, packing pressure, etc.), and b) columns are analyzed with a direct-flow commercially available nano UPLC system. In this presentation we directly compare effects of gradient slope, and column length for two different particles: a novel 1.7 μm bridged-ethyl hybrid (BEH) particle and a 3 μm particle. METHODS Anal. Chem. 2003, 75, 6781-6788 Tetraethoxysilane (TEOS) Bis(triethoxysilyl)ethane (BTEE) + 4 Polyethoxysilane (BPEOS) Si EtO EtO OEt EtO Si EtO EtO CH 2 EtO CH 2 Si OEt OEt OEt Si EtO O CH 2 CH 2 Si O Si EtO OEt Si O O OEt O Si O Si OEt O O OEt Et Et n Bridged Ethanes In Silica Matrix Waters Patented Technology No. 6,686,035 B2 Anal. Chem. 2003, 75, 6781-6788 Tetraethoxysilane (TEOS) Bis(triethoxysilyl)ethane (BTEE) + 4 Polyethoxysilane (BPEOS) Si EtO EtO OEt EtO Si EtO EtO CH 2 EtO CH 2 Si OEt OEt OEt Si EtO O CH 2 CH 2 Si O Si EtO OEt Si O O OEt O Si O Si OEt O O OEt Et Et n Bridged Ethanes In Silica Matrix Waters Patented Technology No. 6,686,035 B2 A very fine human hair, ~ 60 μ m 1.7 μm BEH particles (~ 35 can fit across hair) 10 μm A very fine human hair, ~ 60 μ m 1.7 μm BEH particles (~ 35 can fit across hair) 10 μm Time 10.00 20.00 30.00 40.00 50.00 60.00 psi 0.000 1000.000 2000.000 3000.000 4000.000 5000.000 6000.000 7000.000 8000.000 9000.001 Time 10.00 20.00 30.00 40.00 50.00 60.00 psi 0.000 1000.000 2000.000 3000.000 4000.000 5000.000 6000.000 7000.000 8000.000 9000.001 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 Time 0 100 % J071405HL037 TOF MS ES+ TIC 3.58e4 Figure 4. Reproducibility of six consecutive injections of 100 fmol MassPREP enolase digest with a 180 μm x 20 mm 5 μm Symmetry ® C18 trap column and a 75 μm x 25 cm 1.7 μm BEH C18 analytical column operating at ~9,500 psi. Sample loading/trapping: 2%B at 4 μL/min for 3 min, gradient: 2-60%B in 30 min at 300 nL/min. Average peak width (FWHM) of the peptides for this separation is 4 sec. Figure 5. Overlay of pressure traces for six consecutive injections re- corded on a nano UPLC system, for data shown in Figure 4. Note the ex- cellent reproducibility of gradient delivery for multiple injections recorded over a period of 9 hours. Figure 6. Column performance for 200 fmol 5-protein digest comparing 15 cm long columns packed with (a) 3 μm particles and (b) 1.7 μm parti- cles, gradient: 3-40.6%B in 144 min. Figure 10. MS signal height vs. gradient length for 30 cm, 1.7 μm BEH particle column. The average MS signal height for 20 peptides eluting across the various gradient lengths was normalized to the average value obtained for the 48 min gradient. Peak capacity Gradient length (min) 50 150 250 350 450 550 650 0 100 200 300 400 500 Atlantis 3 μm, 75 μm x 50 cm Atlantis 3 μm, 75 μm x 30 cm Atlantis 3 μm, 75 μm x 15 cm Peak capacity Gradient length (min) 50 150 250 350 450 550 650 0 100 200 300 400 500 BEH 1.7 μm, 75 μm x 30 cm BEH 1.7 μm, 75 μm x 15 cm Peak capacity Gradient length (min) 50 150 250 350 450 550 650 0 100 200 300 400 500 BEH 1.7 μm, 75 μm x 15 cm Atlantis 3 μm, 75 μm x 50 cm Normalized peak height Gradient length (min) 0 20 40 60 80 100 30 100 170 240 310 380 450 LC system: Waters nanoACQUITY UPLC™: • 10,000 psi pressure capability • Direct flow (no splitting) • Accurate nano flow and gradient delivery Columns • 75 μm i.d. (360 μm o.d. fused silica capillary) • Packed with either 3 μm Waters Atlantis ® dC18 or 1.7 μm BEH C18 particles (Figure 1) at 10,000 or 25,000 psi • Nanocolumns were packed to longest length (50 cm for 3 μm , 30 cm for 1.7 μm), tested, cut to specified length(s), and then re-tested to ensure bed consistency. Column temperature: ambient Mobile phase: • Solvent A: 0.1% formic acid • Solvent B: 0.1% formic acid in acetonitrile Gradient conditions: • 3-40.6% B over various durations • Flow rate: 250 nL/min (unless specified otherwise) MS System: Q-Tof micro™ • TOF MS mode • Centroid data • Range: 400-1700 m/z • 1 sec/scan Samples • Waters MassPREP™ yeast enolase or 5-protein digest mixture (bovine serum albumin, yeast enolase, yeast alcohol dehydrogenase, bovine hemoglobin and yeast phosphorylase b) at 100 fmol/μL for each protein Peak capacity measurements • average peak width (FWHM) calculated from XIC’s of 20 peptides taken across the gradient and used to calculate 4 σ • PC = T g /4 σ, where T g is gradient length Figure 2. nanoACQUITY UPLC™ System Figure 8. Gradient length vs. peak capacity plots for 15 and 30 cm length columns packed with 1.7 μm BEH particles. Figure 7. Gradient length vs. peak capacity plots for 15, 30, and 50 cm length columns packed with 3 μm Atlantis particles. Figure 9. Gradient length vs. peak capacity plots comparing performance of a 15 cm, 1.7 μm BEH column with a 50 cm, 3 μm Atlantis column. Figure 3. nanoACQUITY UPLC™ 75 μm x 25 cm column Figure 11. TIC from a nano UPLC separation of 200 fmol each of 5-protein digest for a 30 cm, 1.7 μm BEH column, gradient: 3-40.6%B in 336 min, column backpressure ~ 9,000. Peak capacity for this separation is ~ 550. Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 20.89 20.13 18.67 23.10 21.45 23.94 20.86 18.67 20.13 23.06 21.44 23.91 20.83 20.10 18.65 23.01 21.39 23.87 20.80 18.61 20.06 22.99 21.37 23.83 20.79 20.04 18.59 22.97 21.35 23.82 20.79 18.59 20.04 22.97 21.35 23.82 Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 20.89 20.13 18.67 23.10 21.45 23.94 20.86 18.67 20.13 23.06 21.44 23.91 20.83 20.10 18.65 23.01 21.39 23.87 20.80 18.61 20.06 22.99 21.37 23.83 20.79 20.04 18.59 22.97 21.35 23.82 20.79 18.59 20.04 22.97 21.35 23.82 Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 % 0 100 TOF MS ES+ BPI 9.53e3 20.89 20.13 18.67 23.10 21.45 23.94 E060106AD03 TOF MS ES+ BPI 9.53e3 20.86 18.67 20.13 23.06 21.44 23.91 TOF MS ES+ BPI 9.53e3 20.83 20.10 18.65 23.01 21.39 23.87 TOF MS ES+ BPI 9.53e3 20.80 18.61 20.06 22.99 21.37 23.83 TOF MS ES+ BPI 9.53e3 20.79 20.04 18.59 22.97 21.35 23.82 TOF MS ES+ BPI 9.53e3 20.79 18.59 20.04 22.97 21.35 23.82 Inj #1 Inj #2 Inj #3 Inj #4 Inj #5 Inj #6 • ♣ ♠ ♥ ♦ ◘ std. dev. = 0.04 min • std. dev. = 0.03 min ♠ std. dev. = 0.04 min ♣ std. dev. = 0.04 min ♥ std. dev. = 0.05 min ♦ std. dev. = 0.05 min ◘ Time 20.00 40.00 60.00 80.00 100.00 % 0 100 Time 20.00 40.00 60.00 80.00 100.00 % 0 100 TOF MS ES+ TIC TOF MS ES+ TIC (a) (b) Time 20.00 40.00 60.00 80.00 100.00 % 0 100 Time 20.00 40.00 60.00 80.00 100.00 % 0 100 TOF MS ES+ TIC TOF MS ES+ TIC Time 20.00 40.00 60.00 80.00 100.00 % 0 100 Time 20.00 40.00 60.00 80.00 100.00 % 0 100 TOF MS ES+ TIC TOF MS ES+ TIC (a) (b) PC ~ 350 PC ~ 220 ACKNOWLEDGEMENT We would like to thank Mike Lavallee and Bob Collamati of the Waters Corporation for their technical assistance in packing the columns used for these studies.
Transcript of COMPARISON OF GRADIENT SLOPE, COLUMN LENGTH, AND …We would like to thank Mike Lavallee and Bob...
TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS ©2006 Waters Corporation
OVERVIEW
Purpose: ♦ Directly compare performance of 75 μm i.d. nanocolumns packed
with 1.7 μm versus 3 μm particles for peptide digest separations, investigating effects of column length and gradient length (slope) on peak capacity
Methods: ♦ 10,000 psi nanoACQUITY UPLC™ system coupled to a quadrupole
time-of-flight (Q-Tof) instrument Results: ♦ 1.7 μm particle columns yield higher overall peak capacity gains with
column and gradient length vs. 3 μm particle columns ♦ Shorter (ex. 15 cm) 1.7 μm columns are capable of yielding higher
efficiencies vs. longer (ex. 50 cm) 3 μm columns ♦ There is a tradeoff between increased peak capacity and slight
reduction in MS signal intensity with longer gradient times
COMPARISON OF GRADIENT SLOPE, COLUMN LENGTH, AND PARTICLE SIZES FOR ANALYSIS OF PEPTIDES ON A 10,000 PSI NANOLC/MS/MS PLATFORM
Jeffrey W. Finch, Hongji Liu, Keith Fadgen, Geoff Gerhardt, James P. Murphy, and John C. Gebler Life Sciences R&D, Waters Corporation, Milford, MA, USA
Figure 1. Bridged-ethyl hybrid (BEH) 1.7 μm particle chemistry
References
1. Gilar, M.; Daly, A.E.; Kele, M.; Neue, U.D.; Gebler, J.C. J. Chromatogr A 2004, 1061, 183-192.
2. Shen Y.; Zhang, R.; Moore, R.J.; Kim, J.; Metz, T.O.; Hixson, K.K.; Zhao, R.; Livesay, E.A.; Udeseth, H.A.; Smith, R.D. Anal. Chem. 2005, 77, 3090-3100
3.Tolley, L.; Jorgenson, J.W.; and Moseley, M.A. Anal. Chem. 2001, 73, 2985-2991.
4.Finch J.W.; Stapels, M.D.; Fadgen, K.; Liu, H.; Gerhardt, G.; Murphy, J.P.; Ciavarini, S.; Benevides, C.C.; Gebler, J.C. Proceedings of the 53rd ASMS Conference on Mass Spectrometry, San Antonio, TX, June 2005.
RESULTS
CONCLUSIONS ♦ The nanoACQUITY UPLC system is capable of generating
accurate, reliable gradients at pressures approaching 10,000 psi, with good retention time reproducibility over extended periods of run time
♦ Using a 1.7 μm BEH vs. 3 μm particle for a 15 cm column with a 144 min gradient length results in a 59% increase in peak capacity
♦ For short gradient lengths (24 and 48 min), increasing column length does not significantly increase peak capacity for either particle
♦ The 15 cm, 1.7 μm BEH column yields greater peak capacity measured at the various gradient lengths than the significantly longer 50 cm column packed with 3 μm particles
♦ The 30 cm, 1.7 μm BEH column exhibits the greatest overall increase in peak capacity with gradient length
♦ There is a trade-off between increased separation efficiency and slight reduction in MS signal height with longer gradient lengths
♦ Combining the 1.7 μm BEH particles with longer column lengths and longer gradient lengths can provide greater information for nanoLC/MS/MS analysis of complex proteomic samples
INTRODUCTION The ability to increase nanoLC separation efficiency is highly desired for obtaining as much MS/MS information as possible where amounts of complex proteomics samples are often limited (1). Methods for improving separation efficiency for typical reversed-phase nanoLC separations include using smaller particles, longer columns, and longer (more shallow) gradient times (2,3). The use of smaller particles (< 2 μm) requires a nanoLC system capable of operating at higher backpressure for extended periods of time (4). However, there have been few fundamental studies comparing effects of particle size, column length, and gradient length (time) on peak capacity, for nanocolumns where a) columns are prepared under very tightly-controlled conditions (same column assembly protocol, packing pressure, etc.), and b) columns are analyzed with a direct-flow commercially available nano UPLC system. In this presentation we directly compare effects of gradient slope, and column length for two different particles: a novel 1.7 μm bridged-ethyl hybrid (BEH) particle and a 3 μm particle.
METHODS
Anal. Chem. 2003, 75, 6781-6788
Tetraethoxysilane(TEOS)
Bis(triethoxysilyl)ethane(BTEE)
+4
Polyethoxysilane(BPEOS)
Si
EtO
EtO OEtEtO
Si
EtOEtO
CH2EtO
CH2Si
OEt
OEtOEtSi
EtO
O
CH2 CH2
Si O
Si
EtO
OEt
Si O
O
OEtO
Si
O
Si
OEt
O
OOEt
Et
Et
n
Bridged EthanesIn Silica Matrix
Waters Patented TechnologyNo. 6,686,035 B2
Anal. Chem. 2003, 75, 6781-6788
Tetraethoxysilane(TEOS)
Bis(triethoxysilyl)ethane(BTEE)
+4
Polyethoxysilane(BPEOS)
Si
EtO
EtO OEtEtO
Si
EtOEtO
CH2EtO
CH2Si
OEt
OEtOEtSi
EtO
O
CH2 CH2
Si O
Si
EtO
OEt
Si O
O
OEtO
Si
O
Si
OEt
O
OOEt
Et
Et
n
Bridged EthanesIn Silica Matrix
Waters Patented TechnologyNo. 6,686,035 B2
A very fine human hair, ~ 60 μm
1.7 μm BEH particles(~ 35 can fit across hair)
10 μm
A very fine human hair, ~ 60 μm
1.7 μm BEH particles(~ 35 can fit across hair)
10 μm
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3.58e4
Figure 4. Reproducibility of six consecutive injections of 100 fmol MassPREP enolase digest with a 180 μm x 20 mm 5 μm Symmetry® C18 trap column and a 75 μm x 25 cm 1.7 μm BEH C18 analytical column operating at ~9,500 psi. Sample loading/trapping: 2%B at 4 μL/min for 3 min, gradient: 2-60%B in 30 min at 300 nL/min. Average peak width (FWHM) of the peptides for this separation is 4 sec.
Figure 5. Overlay of pressure traces for six consecutive injections re-corded on a nano UPLC system, for data shown in Figure 4. Note the ex-cellent reproducibility of gradient delivery for multiple injections recorded over a period of 9 hours.
Figure 6. Column performance for 200 fmol 5-protein digest comparing 15 cm long columns packed with (a) 3 μm particles and (b) 1.7 μm parti-cles, gradient: 3-40.6%B in 144 min.
Figure 10. MS signal height vs. gradient length for 30 cm, 1.7 μm BEH particle column. The average MS signal height for 20 peptides eluting across the various gradient lengths was normalized to the average value obtained for the 48 min gradient.
Pea
k ca
paci
ty
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50
150
250
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650
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20
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30 100 170 240 310 380 450
LC system: Waters nanoACQUITY UPLC™: • 10,000 psi pressure capability • Direct flow (no splitting) • Accurate nano flow and gradient delivery
Columns • 75 μm i.d. (360 μm o.d. fused silica capillary) • Packed with either 3 μm Waters Atlantis® dC18 or 1.7 μm BEH
C18 particles (Figure 1) at 10,000 or 25,000 psi • Nanocolumns were packed to longest length (50 cm for 3 μm , 30
cm for 1.7 μm), tested, cut to specified length(s), and then re-tested to ensure bed consistency.
Column temperature: ambient Mobile phase:
• Solvent A: 0.1% formic acid • Solvent B: 0.1% formic acid in acetonitrile
Gradient conditions: • 3-40.6% B over various durations • Flow rate: 250 nL/min (unless specified otherwise)
MS System: Q-Tof micro™
• TOF MS mode • Centroid data • Range: 400-1700 m/z • 1 sec/scan
Samples
• Waters MassPREP™ yeast enolase or 5-protein digest mixture (bovine serum albumin, yeast enolase, yeast alcohol dehydrogenase, bovine hemoglobin and yeast phosphorylase b) at 100 fmol/μL for each protein
Peak capacity measurements • average peak width (FWHM) calculated from XIC’s of 20 peptides
taken across the gradient and used to calculate 4 σ • PC = Tg/4 σ, where Tg is gradient length
Figure 2. nanoACQUITY UPLC™ System
Figure 8. Gradient length vs. peak capacity plots for 15 and 30 cm length columns packed with 1.7 μm BEH particles.
Figure 7. Gradient length vs. peak capacity plots for 15, 30, and 50 cm length columns packed with 3 μm Atlantis particles.
Figure 9. Gradient length vs. peak capacity plots comparing performance of a 15 cm, 1.7 μm BEH column with a 50 cm, 3 μm Atlantis column.
Figure 3. nanoACQUITY UPLC™ 75 μm x 25 cm column
Figure 11. TIC from a nano UPLC separation of 200 fmol each of 5-protein digest for a 30 cm, 1.7 μm BEH column, gradient: 3-40.6%B in 336 min, column backpressure ~ 9,000. Peak capacity for this separation is ~ 550.
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BPI9.53e3
20.8920.1318.67 23.10
21.45 23.9426.06
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9.53e320.86
18.67 20.13 23.0621.4423.91
26.03
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9.53e320.8320.10
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9.53e320.7920.04
18.59 22.9721.3523.82
E060106AD07 TOF MS ES+ BPI
9.53e320.79
18.59 20.0422.9721.35
23.82
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E060106AD03 TOF MS ES+ BPI
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18.61 20.0622.9921.37
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9.53e320.7920.04
18.59 22.9721.3523.82
E060106AD07 TOF MS ES+ BPI
9.53e320.79
18.59 20.0422.9721.35
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0
100
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
%
0
100
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
%
0
100
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
%
0
100E060106AD02 TOF MS ES+
BPI9.53e3
20.8920.1318.67 23.10
21.45 23.9426.06
E060106AD03 TOF MS ES+ BPI
9.53e320.86
18.67 20.13 23.0621.4423.91
26.03
E060106AD04 TOF MS ES+ BPI
9.53e320.8320.10
18.65 23.0121.3923.87
E060106AD05 TOF MS ES+ BPI
9.53e320.80
18.61 20.0622.9921.37
23.83
E060106AD06 TOF MS ES+ BPI
9.53e320.7920.04
18.59 22.9721.3523.82
E060106AD07 TOF MS ES+ BPI
9.53e320.79
18.59 20.0422.9721.35
23.82
Inj #1
Inj #2
Inj #3
Inj #4
Inj #5
Inj #6 • ♣
♠
♥ ♦ ◘
std. dev. = 0.04 min • std. dev. = 0.03 min ♠ std. dev. = 0.04 min ♣ std. dev. = 0.04 min ♥ std. dev. = 0.05 min ♦ std. dev. = 0.05 min ◘
Time20.00 40.00 60.00 80.00 100.00
%
0
100
Time20.00 40.00 60.00 80.00 100.00
%
0
100TOF MS ES+
TIC
TOF MS ES+TIC(a)
(b)
Time20.00 40.00 60.00 80.00 100.00
%
0
100
Time20.00 40.00 60.00 80.00 100.00
%
0
100TOF MS ES+
TIC
TOF MS ES+TIC
Time20.00 40.00 60.00 80.00 100.00
%
0
100
Time20.00 40.00 60.00 80.00 100.00
%
0
100TOF MS ES+
TIC
TOF MS ES+TIC(a)
(b)PC ~ 350
PC ~ 220
ACKNOWLEDGEMENT
We would like to thank Mike Lavallee and Bob Collamati of the Waters Corporation for their technical assistance in packing the columns used for these studies.
matusonl
Text Box
720001736EN
